The development of therapies to eliminate latently infected cells and achieve a functional cure requires standardized assays that reliably and reproducibly assess the size of the latent reservoir. Two approaches have so far been used to estimate the size of the latent reservoir. PCR-based assays are rapid, sensitive, and reproducible, but do not discriminate between replication competent and defective proviruses. By converse, viral outgrowth assays (VOA's) assess the frequency of cells harboring replication competent provirus, but are intensive, variable, and poorly sensitive. An assay that measures the frequency of latently infected cells in a sensitive and reproducible fashion, and that is amenable to a high-throughput, single-cell platform would represent a critical tool for measuring the viral reservoir in future clinical eradication studies. PrimeFlow RNA is a technology that measures RNA expression at the single-cell level by flow cytometry. We have taken advantage of this technique to develop a sensitive, specific, and reproducible assay to detect HIV-1 infected cells. We have developed three HIV-specific probe sets to detect single-spliced, multiple-spliced, and full-length HIV-1 transcripts. By combining two HIV-specific probe sets we identified and enriched infected cells to >90% purity from initial mixtures of 10-100 infected cell per 106 uninfected cells, similar to those observed in vivo. The goal of this application is to validate PrimeFlow RNA for the detection, enumeration, and phenotypic characterization of latently infected cells in clinical samples. This will be accomplished through two specific aims. I Specific Aim 1, we will validate this method by assessing the size of the latent reservoir after ex vivo viral reactivation with latency reversing agents and mitogenic stimuli. We will compare these estimates with the ones obtained by PCR and QVOA. In addition, we will perform longitudinal analyses measuring viral reactivation after in vivo administration of latency reversing agents to HIV-infected individuals enrolled in clinical trials. A significant benefit of PrimeFlow RNA is that it is a flow cytometry-based technology, thus allowing phenotypic characterization as well as sorting of the positive cells. In Specific Aim 2 will take advantage of these aspects. We will sort HIV-infected and uninfected cells from clinical samples by PrimeFlow RNA after ex vivo viral reactivation, which we will use to carry out comparative complete transcriptome analyses. In addition, we will com- bine PrimeFlow RNA with standard flow cytometry to perform comparative analyses of cell surface and intra- cellular markers in HIV-infected and uninfected cells from clinical samples after ex vivo viral reactivation. Therefore the studies proposed in this application will achieve two major goals. First, they will lead to develop a new sensitive technique to measure the size of the latent reservoir. Second, they will generate new critical information about the biology of latently infected cells obtained from clinical samples.